The goal of our current research program is to identify DNA replication initiation sites in mammalian cells with a particular focus on the murine immunoglobulin heavy chain gene (Igh) locus. Our studies identified dramatic changes in the temporal organization of replication with expression. We have suggested that these differences are due to the utilization of different replication initiation sites. We plan to determine the location of replication initiation sites in cells in which the Igh locus is expressed and compare them to sites in which the locus is not expressed. We have demonstrated that the Igh locus, when it is transcriptionally inactive in a murine erythroleukemia cell line, is subdivided into early and late replicated domains. Replication initiates in an early replicated domain, within a 55 kb region downstream of the constant region genes. A single replication fork accomplishes a 400 kb transition from the early replicating to the late replicating domains. This replication fork appears to originate from the last in a cluster of early-activated replicons and proceeds to the first in a cluster of late-activated replicons. The Igh locus could represent a paradigm for many other similar transition regions that are present in mammalian genomes. We now plan to determine whether the initiation region we have identified contains sequence specific replication initiation sites. In the transcriptionally active Igh locus in pre-B cell lines, the transition region is no longer present and the entire locus replicates early in S phase within a narrow interval. We have obtained evidence that at least one additional, pre-B cell-specific, origin is activated in the Igh-C locus when the Igh locus is expressed. We will determine where initiation occurs in the expressed Igh locus. This would be the first tissue specific replication origin(s) identified in mammalian cells. In addition to the powerful techniques we have been using, we are also developing a new approach that combines DNA fiber FISH to identify particular marker sites on DNA molecules and immunofluorescence to determine where replication initiates relative to these marker positions. Thus far, the origin recognition complex (ORC) has not been implicated in the initiation of any mammalian replicon. Identification of replication initiation sites in the Igh locus would allow us to determine whether ORC binds to these sites.